This proposal from Dr. Mark Goldsmith from the Gladstone Institute of Virology is in response to a PA for proposals studying models of HIV disease and AIDS related malignancies. Since HIV infection occurs predominantly within lymphoid tissues in vivo, the investigator proposes to utilize the recently developed "human lymphoid histocultures" or HLH with its' maintenance of tissue viability (up to 21 days), histologic architecture, cell-cell interactions and permissiveness to HIV infection as a model for preclinical investigation. In preliminary work, the investigator and his colleagues have been able to study the molecular and cellular aspects of lymphocyte depletion by HIV. In this application they propose to develop this system to "produce a reliable, quantitative high-resolution and generalized model that is applicable to a variety of important aspects of preclinical investigation including anitviral drug development. In particular, the studies will focus on two essential virologic parameters - replication capacity (fitness) and pathogenic potential (virulence)". In SA 1, the influence of PI resistance mutations on the virulence and fitness properties of HIV will be assessed in HLH based on 1.1. ability of primary PIR and recombinant PIR to deplete CD4 cells in HLH 1.2. ability of PIR virus from patients with isolated virologic failure to deplete CD4 cells. 1.3 competitive assays of wild type and PIR virus in HLH but in the absence of drug to determine relative fitness (assessments will be made phenotypically using GHOST cells and genotypically using HTA). In SA2, HLH will be used to study whether evolution of HIV in vivo is associated with progressively greater virulence or viral fitness: 2.1 whether primary isolates obtained longitudinally exhibit differences in replication potential (viral load) or virulence (CD4 depletion) 2.2 whether naturally attenuated (Nef minus virus from the Sidney cohort) acquire enhanced replication or pathogenic potential through further genomic modifications. In SA 3, the impact of antiviral therapies on HIV fitness, virulence and genetic evolution will be studied in the HLH context: 3.1 define the rate and nature of genetic changes in HIV during replication in HLH with attention to co-receptor specificity in the absence of added selective pressure (assessment by assays with GHOST cells and HTA). 3.2 to determine antiviral therapies such as CXCR4 antagonist T22 or beta chemokines affect env evolution and Protease inhibitors in the case of evolution of protease. The PI has developed several "advanced tools" for the HLH system including the ability to FACS based on CXCR4 and CCR5 expression to distinguish individual effects on these cells types and kinetic (time course) evaluations of cell depletion.